FR2707664A1 - Viral vectors and use in gene therapy - Google Patents

Abstract

The present invention relates to new viral vectors derived from adenoviruses, to their preparation and to their use in gene therapy.

Description

The present invention: VIRAL VECTORS AND USE IN T-ERAPr GENIOUE

  relates to new viral vectors, their preparation and their use in gene therapy. It also relates to pharmaceutical compositions containing said viral vectors. More particularly, the present invention relates to recombinant adenoviruses as vectors for therapy

gene.

  Gene therapy consists of correcting a deficiency or an abnormality (mutation, aberrant expression, etc.) by introducing genetic information into the affected cell or organ. This genetic information can be introduced either in vitro into a cell extracted from the organism, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, different techniques exist, among which various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J.Lvirol. 1 (1967) 891), of DNA and proteins nuclear (Kaneda et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), use of liposomes (Fraley et al., J.] iol.Chem 255 (1980) 10431), etc. More recently, the use of viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques. In this regard, different viruses have been tested for their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), the HSV virus,

  adeno-associated viruses, and adenoviruses.

  Among these viruses, adenoviruses have certain properties of interest for use in gene therapy. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, do not integrate into the genome of the infected cell, and have not been associated to date with significant pathologies

in humans.

  Adenoviruses are linear double-stranded DNA viruses approximately 36 kb in size. Their genome notably includes a reverse inverted sequence (ITR) at their end, an encapsidation sequence, early genes and late genes (Cffigure 1). The main early genes are the El (Ela and Elb), E2, E3 and

  E4. The main late genes are the LI to L5 genes.

  Given the properties of the adenoviruses mentioned above, these have already been used for gene transfer in vivo. For this purpose, different vectors

  adenovirus derivatives were prepared, incorporating different genes (B13-gal, OTC, ac-

  1AT, cytokines, etc.). In each of these constructions, the adenovirus was modified so as to render it incapable of replication in the infected cell. Thus, the constructions described in the prior art are adenoviruses deleted from the El (Ela and / or Elb) and possibly E3 regions into which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195 ; Gosh-Choudhury et al., Gene 50 (1986) 161). However, the vectors described in the prior art have many drawbacks which limit their use in gene therapy. In particular, all of these vectors contain numerous viral genes whose expression in vivo is undesirable in the context of gene therapy. In addition, these vectors do not allow the incorporation of very large DNA fragments, which

  may be required for some applications.

  The present invention overcomes these drawbacks. The present invention indeed describes recombinant adenoviruses for gene therapy, capable of efficiently transferring DNA (up to 30 kb) in vivo, of expressing this DNA at high levels and in a stable manner in vivo, limiting any risk of viral protein production, virus transmission, pathogenicity, etc. In particular, it has been found that it is possible to considerably reduce the size of the

  adenovirus genome, without preventing the formation of an encapsidated viral particle.

  This is surprising since it has been observed in the case of other viruses, for example retroviruses, that certain sequences distributed along the genome are necessary for effective packaging of the viral particles. As a result, the production of vectors having large internal deletions was greatly limited. The present invention also shows that the deletion of most of the viral genes does not prevent the formation of such a viral particle either. In addition, the recombinant adenoviruses thus obtained retain, despite significant modifications to their genomic structure, their advantageous properties of high infectivity, stability in vivo, etc. The vectors of the invention are particularly advantageous since they allow incorporation of very large desired DNA sequences. It is thus possible to insert a gene with a length greater than 30 kb. This is particularly interesting for certain pathologies whose treatment requires the co-expression of several genes, or the expression of very large genes. So for example, in the case of muscular dystrophy, it was not possible until now to transfer the cDNA corresponding to the native gene responsible for this pathology

  (dystrophin gene) due to its large size (14 kb).

  The vectors of the invention are also very advantageous since they have very few functional viral regions and therefore, the risks inherent in the use of viruses as vectors in gene therapy such as immunogenicity, pathogenicity, transmission, replication, recombination, etc.

  are greatly reduced or even eliminated.

  The present invention thus provides particularly viral vectors

  suitable for the transfer and expression in vivo of desired DNA sequences.

  A first object of the present invention therefore relates to a defective recombinant adenovirus comprising: - the TIR sequences, - a sequence allowing encapsidation, - a heterologous DNA sequence, and in which: - the E1 gene is non-functional and

  - at least one of the E2, E4, L1-L5 genes is non-functional.

  Within the meaning of the present invention, the term “defective adenovirus designates a

  adenovirus unable to replicate autonomously in the target cell.

  Generally, the genome of the defective adenoviruses according to the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the

heterologous DNA sequence.

  The inverted repeat sequences (ITR) constitute the origin of replication of adenoviruses. They are located at the 3 ′ and 5 ′ ends of the viral genome (cf. FIG. 1), from which they can be easily isolated according to the conventional techniques of molecular biology known to those skilled in the art. The nucleotide sequence of the ITR sequences of human adenoviruses (in particular the Ad2 and Ad5 serotypes) is described in

  the literature, as well as canine adenoviruses (in particular CAV1 and CAV2).

  For the Ad5 adenovirus for example, the left ITR sequence corresponds to the

  region comprising nucleotides 1 to 103 of the genome.

  The packaging sequence (also called Psi sequence) is necessary for packaging the viral DNA. This region must therefore be present to allow the preparation of defective recombinant adenoviruses according to the invention. The packaging sequence is located in the genome of the adenoviruses, between 'left ITIR (5') and the E1 gene (see FIG. 1). It can be isolated or artificially synthesized by conventional molecular biology techniques. The nucleotide sequence of the packaging sequence of human adenoviruses (in particular the Ad2 and Ad5 serotypes) is described in the literature, as well as canine adenoviruses (in particular CAV1 and CAV2). Regarding AdS adenovirus for example, the packaging sequence

  corresponds to the region comprising nucleotides 194 to 358 of the genome.

  There are different serotypes of adenovirus, the structure and properties of which vary somewhat. Nevertheless, these viruses have a comparable genetic organization, and the lessons described in the present application can be

  easily reproduced by a person skilled in the art for any type of adenovirus.

  The adenoviruses of the invention can be of human, animal, or

mixed (human and animal).

  As regards adenoviruses of human origin, it is preferred to use those classified in group C. More preferably, among the various serotypes of human adenovirus, it is preferred to use, within the framework of the present invention, adenoviruses of

type 2 or 5 (Ad 2 or Ad 5).

  As indicated above, the adenoviruses of the invention can also be of animal origin, or contain sequences derived from adenoviruses of animal origin. The Applicant has indeed shown that adenoviruses of animal origin are capable of infecting human cells with great efficiency, and that they are unable to propagate in the human cells in which they have been tested (see request FR 93 05954). The Applicant has also shown that adenoviruses of animal origin are in no way trans-complemented by adenoviruses of human origin, which eliminates any risk of recombination and of propagation in vivo, in the presence of a human adenovirus, which can lead to the formation of an infectious particle. The use of adenoviruses or adenovirus regions of animal origin is therefore particularly advantageous since the risks inherent in the use of viruses

  as vectors in gene therapy are even weaker.

  The adenoviruses of animal origin which can be used in the context of the present invention can be of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or else simienne (example: after-sales service). More particularly, among avian adenoviruses, there may be mentioned serotypes 1 to 10 accessible to rATCC, such as for example the strains Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR-827) , IBH-2A (ATCC VR-828), J2-A (ATCC VR- 829), T8-A (ATCC VR-830), K-11 (ATCC VR-921) or the strains referenced ATCC VR-831 to 835. Among the bovine adenoviruses, it is possible to use the various known serotypes, and in particular those available at ATCC (types i to 8) under the references ATCC VR-313, 314, 639-642, 768 and 769. It is possible to use

  also mention the murine adenoviruses FL (ATCC VR-550) and E20308 (ATCC VR-

  528), sheep adenovirus type 5 (ATCC VR-1343), or type 6 (ATCC VR-1340); porcine radenovirus 5359), or simian adenoviruses such as in particular adenoviruses referenced to 'ATCC under the numbers VR-591-594, 941-943, 195-203, etc. Preferably, among the various adenoviruses of animal origin, adenoviruses or regions of adenoviruses of canine origin are used in the context of the invention, and in particular all the strains of adenovirus CAV2 [manhattan or A26 / 61 strain (ATCC VR -800) for example]. Canine adenoviruses have been the subject of numerous structural studies. Thus, complete restriction maps of

  CAV1 and CAV2 adenoviruses have been described in the prior art (Spibey et al., J. Gen.

  Virol. 70 (1989) 165), and the Ela, E3 genes as well as the ITR sequences have been cloned and sequenced (see in particular Spibey and aL, Virus Res. 14 (1989) 241; Linné, Virus

Res. 23 (1992) 119, WO 91/11525).

  As indicated above, the adenoviruses of the present invention comprise a heterologous DNA sequence. The heterologous DNA sequence designates any DNA sequence introduced into the recombinant virus, the transfer and / or

  the expression in the target cell is sought.

  In particular, the heterologous DNA sequence may contain one or more therapeutic genes and / or one or more genes coding for antigenic peptides. The therapeutic genes which can thus be transferred are any gene whose transcription and possibly translation into the target cell generate

  products having a therapeutic effect.

  They may in particular be genes coding for protein products having a therapeutic effect. The protein product thus coded can be a protein, a peptide, an amino acid, etc. This protein product can be homologous with respect to the target cell (that is to say a product which is normally expressed in the target cell when the latter presents no pathology). In this case, the expression of a protein makes it possible, for example, to compensate for an insufficient expression in the cell or re-expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein. The therapeutic gene can also code for a mutant of a cellular protein, having increased stability, modified activity,

  etc. The protein product can also be heterologous towards the target cell.

  In this case, an expressed protein can for example supplement or provide a

  deficient activity in the cell allowing it to fight against a pathology.

  Among the therapeutic products within the meaning of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines: interleukldnes, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NTS, etc; apolipoproteins: ApoAI, ApoAIV, ApoE, etc (FR 93 05125), dystrophin or a minidystrophin (FR 9111947), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc (FR 93 04745) , genes coding for factors involved in coagulation: Factors VI, VIII IX, etc. The therapeutic gene can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNA. Such sequences can for example be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP

140,308.

  As indicated above, the heterologous DNA sequence may also contain one or more genes coding for an antigenic peptide capable of generating an immune response in humans. In this particular mode of implementation, the invention therefore allows the production of vaccines making it possible to immunize humans, in particular against microorganisms or viruses. They may in particular be antigenic peptides specific for the epstein barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, or else

  specific for tumors (EP 259 212).

  Generally, the heterologous DNA sequence also comprises sequences allowing re-expression of the therapeutic gene and / or of the gene coding for the antigenic peptide in the infected cell. These may be sequences which are naturally responsible for re-expression of the gene considered when these sequences are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which one wishes to infect. Likewise, they may be promoter sequences originating from the genome of a virus, including the radenovirus used. In this regard, mention may be made, for example, of the promoters of the E1A, MLP, CMV, RSV, etc. genes. In addition, these expression sequences can be modified by adding activation, regulation sequences, etc. Furthermore, when the inserted gene does not contain expression sequences, it can be inserted into the genome of the defective virus downstream

of such a sequence.

  Furthermore, the heterologous DNA sequence may also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or a signal sequence

artificial.

  As indicated above, the vectors of the invention have at least one of the non-functional E2, E4, L1-L5 genes. The viral gene considered can be made non-functional by any technique known to a person skilled in the art, and in particular by deletion, substitution, deletion, or addition of one or more bases in the gene or genes considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or alternatively

  by treatment with mutagens.

  Among the mutagenic agents, mention may be made, for example, of physical agents such as energetic radiation (X-rays, ultra violet, etc.), or chemical agents capable of reacting with different functional groups of the bases of

  DNA, and for example alkylating agents [ethylmethane sulfonate (EMS), N-methyl-

  N-nitro-N-nitrosoguanidine, N-nitroquinoline-l-oxide (NQO)], bialkylating agents, intercalating agents, etc. By deletion is meant in the sense of the invention any deletion of the gene considered. It may especially be all or part of the coding region of said gene, and / or all or part of the region promoting the transcription of said gene. The removal can be carried out by digestion using appropriate restriction enzymes, then ligation, according to conventional techniques of molecular biology, as well

as illustrated in the examples.

  Genetic changes can also be obtained by disruption

  gene, for example according to the protocol initially described by Rothstein [Meth.

  Enzymol. 101 (1983) 202]. In this case, all or part of the coding sequence is preferably disturbed to allow replacement, by homologous recombination, of the genomic sequence with a non-functional or mutant sequence. The said genetic modification (s) may be located in the coding part of the gene concerned, or outside the coding region, and for example in the regions responsible for the expression and / or transcriptional regulation of said genes. The non-functional character of said genes can therefore be manifested by the production of an inactive protein due to structural or conformational modifications, by absence of production, by the production of a protein having an altered activity, or by the production of the natural protein at one level

  attenuated or according to a desired regulation mode.

  In addition, certain alterations such as point mutations are by their nature capable of being corrected or attenuated by cellular mechanisms. Such genetic alterations are therefore of limited interest at the industrial level. It is therefore particularly preferred that the non-functional character be perfectly stable

  segregated and / or non-reversible.

  Preferably, the gene is non-functional due to a partial deletion

or total.

  Preferably, the defective recombinant adenoviruses of the invention are

  lacking late adenovirus genes.

  A particularly advantageous embodiment of the invention consists of a defective recombinant adenovirus comprising: - the ITR sequences, - a sequence allowing recapsidation, - a heterologous DNA sequence, and

  - a region carrying the gene or part of the E2 gene.

  Another particularly advantageous embodiment of the invention consists of a defective recombinant adenovirus comprising: - the ITR sequences, - a sequence allowing recapsidation, - a heterologous DNA sequence, and

  - a region carrying the gene or part of the E4 gene.

  Still in a particularly advantageous mode, the vectors of the invention also have a functional E3 gene under the control of a heterologous promoter. More preferably, the vectors have a part of the E3 gene

  allowing reexpression of the protein gpl9K.

  The defective recombinant adenoviruses according to the invention can be prepared

different ways.

  A first method consists in transfecting the DNA of the defective recombinant virus prepared in vitro (either by ligation or in the form of a plasmid) in a competent cell line, that is to say carrying in trans all the functions necessary for the complementation of the defective virus These functions are preferably integrated into the genome of the cell, which makes it possible to avoid the risks of recombination, and confers increased stability to the cell line. The preparation of such lines

  cellular is described in the examples.

  A second approach consists in co-transfecting into a suitable cell line rDNA of the defective recombinant virus prepared in vitro (either by ligation or in the form of a plasmid) and rDNA of a helper virus. According to this method, it is not necessary to have a competent cell line capable of complementing all the defective functions of the recombinant adenovirus. Part of these functions is indeed complemented by the helper virus. This helper virus must itself be defective and the cell line carries in trans the functions necessary for its complementation. The preparation of defective recombinant adenoviruses of the invention

  according to this method is also illustrated in the examples.

  Among the cell lines which can be used in the context of this second approach, mention may in particular be made of the human embryonic kidney line 293,

  KB cells, Hela cells, MDCK, GHK, etc. (see examples).

  Then, the vectors which have multiplied are recovered, purified and amplified according to conventional techniques of molecular biology. The present invention therefore also relates to cell lines infectable by adenoviruses, comprising, integrated into their genome, the functions necessary for the complementation of a defective recombinant adenovirus as described above. In particular, it relates to cell lines comprising, integrated into their genome, the El and E2 regions (in particular the region coding for the

  protein 72K), and / or E4 and / or the glucocorticoid receptor gene.

  Preferably, these lines are obtained from line 293 or gm DBP6.

  The present invention also relates to any pharmaceutical composition comprising one or more defective recombinant adenoviruses as described above. The pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the pharmaceutical composition contains pharmaceutically acceptable vehicles for an injectable formulation. They may in particular be saline solutions (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by adding, as appropriate, sterilized water or physiological saline, allow the constitution of

injectable solutions.

  The doses of virus used for the injection can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfiu / ml, and preferably 106 to 1010 pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 5 days, the number of plaques of infected cells. Techniques for determining the pfu titer of a viral solution

  are well documented in the literature.

  Depending on the heterologous DNA sequence inserted, the adenoviruses of the invention can be used for the treatment or prevention of many pathologies, including genetic diseases (dystrophy, cystic fibrosis, etc.), neurogenerative diseases (alzheimer, parkinson, ALS , etc.), cancers, pathologies linked to coagulation disorders or dyslipoproteinemias, pathologies linked to viral infections (hepatitis, AIDS, etc.), etc. The present invention will be more fully described with the aid of the examples.

  which follow, which should be considered as illustrative and not limiting.

  Legend of the fairs Figure 1: Genetic organization of Ad5 radenovirus. The complete sequence of Ad5 is available on the database and allows a person skilled in the art to select or

  to create any restriction site, and thus to isolate any region of the genome.

  Figure 2: Restriction map of the CAV2 adenovirus strain Manhattan (from

Spibey et al supra).

  Figure 3: Construction of defective viruses of the invention by ligation.

  Figure 4: Construction of a recombinant virus carrying the E4 gene.

  Figure 5: Construction of a recombinant virus carrying the E2 gene.

  General molecular biology techniques The methods conventionally used in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification DNA fragments by electroelution, phenol or phenol-chloroform protein extraction, DNA precipitation in saline medium with rethanol or isopropanol, transformation in Escherichia coli, etc. are well known skilled in the art and are abundantly described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F.M. et al. (eds), "Current Protocols in

  Molecular Biology ", John Wiley & Sons, New York, 1987].

  Plasmids of the pBR322, pUC and phage type M13 sede are

  of commercial origin (Bethesda Research Laboratories).

  For the ligations, the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol then incubated in the presence of DNA.

  phage T4 ligase (Biolabs) according to the supplier's recommendations.

  The filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications. The destruction of the prominent 3 'ends is carried out in the presence of rDNA Polymerase from phage T4 (Biolabs) used according to the manufacturer's recommendations. Destruction of the prominent 5 'ends is effected by treatment

formed by nuclease S1.

  Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13

  (1985) 8749-8764] using the kit distributed by Amersham.

  Enzymatic amplification of DNA fragments by the so-called PCR technique

  [: olymérase-catalyzed Chair eaction, Saiki R.K. et ai., Science 2Q (1985) 1350-

  1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the

manufacturer's specifications.

  Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 7.4 (1977) 5463-5467] in

  using the kit distributed by Amersham.

  Cell lines used In the following examples, the following cell lines have or can be used: - Human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59). This line contains in particular, integrated into its genome, the part

  left of the human adenovirus AdS genome (12%).

  - Human cell line KB: From a human epidermal carcinoma, this line is available at the ATCC (ref. CCL17) as well as the conditions allowing

his culture.

  - Human cell line Hela: From a human repithelium carcinoma, this line is available at r'ATCC (ref. CCL2) as well as the conditions

allowing its cultivation.

  - MDCK canine cell line: The culture conditions for MDCK cells have been described in particular by Macatney et al., Science 44 (1988) 9.

  - gmin DBP6 cell line (Brough et al., Virology 190 (1992) 624).

  This line consists of Hela cells carrying the E2 adenovirus gene under the

MMTR LTR control.

Example i

  This example demonstrates the feasibility of a recombinant adenovirus lacking the essentials of viral genes For this, a series of deletion mutants in

  adenovirus was constructed by in vitro ligation, and each of these mutants was co-

  transfected with helper virus in KB cells. These cells do not allow the propagation of viruses defective for E1, the transcomplementation relates to the E1 region. The various deletion mutants were prepared from the Ad5 adenovirus by digestion then ligation in vitro. For this, the Ad5 viral DNA is isolated according to the technique described by Lipp et al. (J. Virol. 63 (1989) 5133), subjected to digestion in the presence of different restriction enzymes (cf. FIG. 3), then the digestion product is ligated in the presence of T4 DNA ligase. The size of the various deletion mutants is then checked on 0.8% SDS agarose gel. These mutants are then mapped (see FIG. 3). These different mutants include the following regions: mtl: Ligation between the fragments of Ad5 0-20642 (SauI) and (SauI) 33797-35935 mt2: Ligation between the fragments of Ad5 0- 19549 (NdeI) and (NdeI) 31089 -35935 mt3: Ligation between Ad5 fragments 0-10754 (AatII) and (Aatfl) 25915-35935 mt4: Ligation between Ad5 fragments 0-113 1 (Mlul) and (MluI) 24392-35935 mt5: Ligation between Ad5 fragments 0-9462 (Sall) and (XhoI) 29791-35935 mt6: Ligation between Ad5 fragments 0-5788 (XhoI) and (XhoI) 29791-35935 mt7: Ligation between Ad5 fragments 0-3665 (SphI) and (Sphli) 31224-35935 Each of the mutants prepared above was co-transfected with viral DNA from Ad. RSVBGal (Stratford-Perricaudet et al., J. Clin.Invest. 90 (1992 ) 626) in KB cells, in the presence of calcium phosphate. The cells were harvested 8 days after transfection, and the culture supernatants were harvested and then amplified on KB cells until stocks of 50 dishes were obtained for each transfection. From each sample, the episomal DNA was isolated and separated on a cesium chloride gradient. Two distinct bands of virus were observed in each case, sampled and analyzed. The heaviest corresponds to the viral DNA of Ad.RSVBGal, and the lightest corresponds to the DNA of the recombinant virus generated by ligation (FIG. 3). The title

  obtained for the latter is approximately 108 pfu / ml.

  A second series of deletion mutants in radenoviruses was constructed by in vitro ligation according to the same methodology. These different mutants include the following regions:

  mt8: Ligation between fragments 0-4623 (ApaI) of Ad RSVI3Gal and (ApaI) 31909-

35935 of Ad5.

  mt9: Ligation between fragments 0-10178 (BglID of RSVBGal Ad and (BamHI) 21562-

35935 of Ad5.

  These mutants, carrying the LacZ gene under the control of the RSV virus LTR promoter, are then co-transfected in 293 cells in the presence of the viral DNA of

  H2dl808 (Weinberg et al., J. Virol. 57 (1986) 833), which is deleted from the E4 region.

  According to this second technique, the transcomplementation relates to E4 and no longer to El. This technique thus makes it possible to generate, as described above, viruses

  recombinants having only the E4 region as a viral gene.

Example 2

  This example describes the preparation of defective recombinant adenoviruses according to the invention by co-transfection, with a helper virus, of the DNA of the recombinant virus.

incorporated into a plasmid.

  For this, a plasmid carrying the adjoining IT1Ks of the AdS, the packaging sequence, the E4 gene under the control of its own promoter and, as a heterologous gene, the LacZ gene under the control of the LTR promoter of the RSV virus was constructed ( figure 4). This plasmid, designated pE4Gal, was obtained by cloning and ligation of the following fragments (see FIG. 4): Ind11-Sac11 fragment from the plasmid pFG144 (Graham et al, EMBO J. 8 (1989) 2077). This fragment carries the ITR sequences of Ad5 head to tail and the

  packaging sequence: Hindif fragment (34920) -Sacii (352).

  - Ad5 fragment between the SacIl sites (located at the base pair 3827) and PstI (located at the base pair 4245); - fragment of pSP 72 (Promega) between the PstI (bp 32) and SalI (bp 34) sites;

  s - XhoI-XbaI fragment of the plasmid pAdLTR GaIX described in Stratford-

  Perricaudet et al (JCI 90 (1992) 626). This fragment carries the Lacz gene under control

of the RSV virus LTR.

  - XbaI fragment (bp 40) - NdeI (bp 2379) of the plasmid pSP 72; - NdeI fragment (bp 31089) - HindIII (bp 34930) of Ad5. This fragment located in the right end of the r'Ad5 genome contains the E4 region under the control of its own promoter. It was cloned at the NdeI sites (2379) of the plasmid

  pSP 72 and Hindill of the first fragment.

  This plasmid was obtained by cloning of the different fragments in the regions indicated of the plasmid pSP 72. It is understood that equivalent fragments

  can be obtained by those skilled in the art from other sources.

  The plasmid pE4Gal is then co-transfected with the DNA of the H2dl808 virus in the 293 cells in the presence of calcium phosphate. The recombinant virus is then prepared as described in example 1. This virus carries, as the only viral gene, the E4 gene of the Ad5 adenovirus (FIG. 4). Its genome is about 12 kb in size,

  which allows the insertion of very large heterologous DNA (up to 20 kb).

  Thus, a person skilled in the art can easily replace the LacZ gene with any other therapeutic gene such as those mentioned above. Furthermore, this virus contains certain sequences originating from the plasmid pSP 72, which can be eliminated by the

  classical molecular biology techniques if necessary.

Example 3

  This example describes the preparation of another defective recombinant adenovirus according to the invention by co-transfection, with a helper virus, of the DNA of the virus.

  recombinant incorporated into a plasmid.

  For this, a plasmid carrying the adjoining ITRs of rAd5, the packaging sequence, the E2 gene of Ad2 under the control of its own promoter and, as a heterologous gene, the LacZ gene under the control of the LTR promoter of the RSV virus was constructed (Figure 5). This plasmid, designated pE2Gal, was obtained by cloning and ligation of the following fragments (see FIG. 5): HindIII-SacIl fragment derived from the plasmid pFG144 (Graham et al, EMBO J. 8 (1989) 2077). This fragment carries the 1TR sequences of AdS head to tail and the packaging sequence: HindI fragment (34920) -SacII (352). He was cloned, with

  the following fragment, at the HindIfI (16) - PstI (32) sites of the plasmid pSP 72.

  fragment of rAd5 comprised between the Sac11 sites (located at the base pair 3827) and PstI sites (located at the base pair 4245). This fragment was cloned at the SacHI site of the previous fragment and at the PstI site (32)

of the plasmid pSP 72.

  - fragment of pSP 72 (Promega) between the PstI (bp 32) and SalI (bp 34) sites;

  - XhoI-XbaI fragment of the plasmid pAdLTR GafIX described in Stratford-

  Perricaudet et al (ICI 90 (1992) 626). This fragment carries the LacZ gene under the control of the LTR of the RSV virus. It was cloned at the SalI (34) and XbaI sites of the plasmid

pSP 72.

  - fragment of pSP 72 (Promega) comprised between the XbaI (pb 34) and BamH sites [(pb 46); - BamHI fragment (bp 21606) - SmaI (bp 27339) of Ad2. This fragment of the Ad2 genome contains the E2 region under the control of its own promoter. He was

  cloned at the BamiI (46) and EcoRV sites of the plasmid pSP 72.

  - EcoRV (bp 81) - HindiI (bp 16) fragment of the plasmid pSP 72.

  This plasmid was obtained by cloning of the different fragments in the regions indicated of the plasmid pSP 72. It is understood that equivalent fragments

  can be obtained by those skilled in the art from other sources.

  The pE2Gal plasmid is then co-transfected with the DNA of the H2dl802 virus lacking the E2 region (Rice et al. J. Virol. 56 (1985) 767) in 293 cells, in the presence of calcium phosphate. The recombinant virus is then prepared as described in example 1. This virus carries, as the only viral gene, the E2 gene of the adenovirus Ad2 (FIG. 5). Its genome is approximately 12 kb in size, which allows the insertion of very large heterologous DNA (up to 20 kb). Thus, a person skilled in the art can easily replace the LacZ gene with any other therapeutic gene such as those mentioned above. Furthermore, this virus contains certain sequences originating from the intermediate plasmid, which can be eliminated by techniques

  molecular biology classics if necessary.

  F ^^ ipe 4 This example describes the construction of complementary cell lines for the E1, E2 and / or E4 regions of the adenoviruses. These lines allow the construction of recombinant adenoviruses according to the invention deleted for these regions, without using a helper virus. These viruses are obtained by recombination in vivo, and

  may contain important heterologous sequences.

  In the cell lines described, the E2 and E4 regions, potentially cytotoxic, are placed under the control of an inducible promoter: the LTR of MMTV (Pharmacia), which is induced by dexamethasone. It is understood that other promoters can be used, and in particular variants of the LTR of MMTV carrying, for example, heterologous regulatory regions ("enhancer" region in particular). The invention lines are constructed by transfection of the corresponding cells, in the presence of calcium phosphate, with a DNA fragment carrying the indicated genes (adenovirus regions and / or glucocorticoid receptor gene) under the control of a promoter of transcription and a terminator (polyad4nylation site). The terminator can be either the natural terminator of the transfected gene, or a different terminator, for example the terminator of the SV40 virus early messenger. Advantageously, the DNA fri-agment also carries a gene allowing the selection of transformed cells, and for example the gene for resistance to geneticin. The resistance gene can

  also be carried by a different DNA fragment, co-transfected with the first.

  After transfection, the transformed cells are selected and their DNA

  is analyzed to verify the integration of the DNA fragment into the genome.

  This technique makes it possible to obtain the following cell lines: 1. 293 cells possessing the 72K gene of the E2 region of Ad5 under the control of the LTR of MM-TV; 2. 293 cells possessing the 72K gene from the E2 region of Ad5 under the control of the LTR of MMTV and the glucocorticoid receptor gene; 3. 293 cells possessing the 72K gene of the E2 region of Ad5 under the control of the LTR of MMTV and the E4 region under the control of the LTR of MMTV; 4. 293 cells possessing the 72K gene of the E2 region of Ad5 under the control of the LTR of MMIV, the E4 region under the control of the LTR of MMTV, and the gene for the glucocorticoid receptor; 5. 293 cells possessing the E4 region under the control of the LTR of MMTV; 6. 293 cells possessing the E4 region under the control of the MMIV LTR and the glucocorticoid receptor gene; 7. gm DBP6 cells having the E1A and E1B regions under the control of their own promoter, 8. gm DBP6 cells having the EIA and E1B regions under the control of their

  own promoter and the E4 region under the control of the LTR of MMTV.

Claims (26)

  1. Defective recombinant adenovirus comprising: - the ITR sequences, - a sequence allowing the packaging, - a heterologous DNA sequence,   and in which the E1 gene and at least one of the E2, E4, L1-L5 genes is non-functional.   2. Adenovirus according to claim 1 characterized in that it is of origin human, animal, or mixed.   3. Adenovirus according to claim 2 characterized in that the adenoviruses of human origin are chosen from those classified in group C, preferably   among adenoviruses type 2 or 5 (Ad 2 or Ad 5).   4. Adenovirus according to claim 2 characterized in that the adenoviruses of animal origin are chosen from adenoviruses of canine, bovine, murine origin,   ovine, porcine, avian and sinian.   5. Adenovirus according to one of the preceding claims, characterized in that   that it lacks late genes.   6. Adenovirus according to claim 1 characterized in that it comprises: the ITR sequences, - a sequence allowing recapsidation, - a heterologous DNA sequence, and   - a region carrying the gene or part of the E2 gene.   7. Adenovirus according to claim 1 characterized in that it comprises: the ITR sequences, - a sequence allowing the encapsidation, - a heterologous DNA sequence, and   - a region carrying the gene or part of the E4 gene.   8. Adenovirus according to one of the preceding claims, characterized in that   that it further comprises a functional E3 gene under the control of a heterologous promoter.   9. Adenovirus according to one of the preceding claims, characterized in that   that the heterologous DNA sequence contains one or more therapeutic genes   and / or one or more genes coding for antigenic peptides.   10. Adenovirus according to claim 9 characterized in that the therapeutic gene is chosen from the genes coding for enzymes, blood derivatives, hormones, lymphokines (interleukins, interferons, TNF, etc.), growth factors, neurotransmitter dies or their synthetic precursors or enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc.), apolipoproteins (ApoAI, ApoAIV, ApoE, etc.), dystrophin or a minidystrophin , tumor suppressor genes or genes encoding for   factors involved in coagulation (Factors VII, VIII, IX, etc.).   He. Adenovirus according to Claim 9, characterized in that the therapeutic gene is an antisense gene or sequence, the expression of which in the cell   target allows to control gene expression or transcription of cellular mRNA.   12. Adenovirus according to claim 9 characterized in that the gene encodes an antigenic peptide capable of generating in humans an immune response   against microorganisms or viruses.   13. Adenovirus according to claim 12 characterized in that the gene codes for an antigenic peptide specific for the epstein barr virus, the H1V virus, the virus   hepatitis B, the pseudo-rabies virus, or specific for tumors.   14. Adenovirus according to one of the preceding claims, characterized in that   that the heterologous DNA sequence also includes sequences allowing the expression of the therapeutic gene and / or of the gene coding for the antigenic peptide in the infected cell.   15. Adenovirus according to one of the preceding claims, characterized in that   that the heterologous DNA sequence comprises, upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the pathways secretion of the target cell.   16. Cell line infectable by an adenovirus comprising, integrated into its genome, the functions necessary for the complementation of an adenovirus   defective recombinant according to one of claims 1 to 15.   17. Cell line according to claim 16 characterized in that it   has in its genome at least the E1 and E2 genes of an adenovirus.   18. Cell line according to claim 17 characterized in that it   also contains the E4 gene from an adenovirus.   19. Cell line according to claim 16 characterized in that it   contains in its genome at least the E1 and E4 genes of an adenovirus.   20. Cell line according to claims 17 to 19 characterized in that it   also contains the glucocorticoid receptor gene.   21. Cell line according to Claims 17 to 20, characterized in that   the E2 and E4 genes are placed under the control of an inducible promoter.   22. Cell line according to claim 21 characterized in that the   inducible promoter is the LTR promoter of MMTV.   23. Cell line according to claims 17 to 22, characterized in that the   E2 gene codes for the 72K protein.   24. Cell line according to claims 16 to 23, characterized in that it   is obtained from line 293.   25. Pharmaceutical composition comprising at least one adenovirus   defective recombinant according to any of claims 1 to 15.   26. The pharmaceutical composition according to claim 25 comprising a   Recombinant adenovirus according to one of claims 5 to 7.   27. Pharmaceutical composition according to claims 25 or 26   comprising a pharmaceutically acceptable vehicle for a formulation injectable.